BOT-605 Asif Saeed course of advance gentics.pptx

This document contains advance genetics and vectors in Genetic engineering.

1. Vectors for Gene Cloning:Plasmids and Bacteriophages Kaleem Jaffar 2020-ag-9140 Vectors In Genetic Engineering

2. Vectors for Gene Cloning:Plasmids and Bacteriophages Plasmids: Basic features of plasmids : Plasmids are circular DNA molecules that can replicate independently in bacterial cells. They often carry genes that give the host bacterium useful traits, like antibiotic resistance. In the lab, we can use antibiotic resistance as a marker to select bacteria with a specific plasmid. Plasmids have an origin of replication that allows them to multiply independently of the bacterial chromosome. . Vectors for Gene Cloning:Plasmids and Bacteriophages

3. Smaller plasmids use the host cell's replicative enzymes, while larger ones have their own specialized enzymes . Some plasmids can integrate into the bacterial chromosome and exist as independent elements. This integration is also seen in bacteriophage chromosomes. Size and copy number : Plasmid size: Smaller plasmids (<10kb) are best for cloning. Plasmid range: They can vary from 1.0 kb to over 250 kb. .

4. Copy number: It's the number of plasmid molecules in a bacterial cell. Factors: The factors controlling copy number are not well understood Useful cloning vector: It should be present in multiple copies for high DNA yields. Conjugaton and compatibility: Plasmids in bacteria can be conjugative or non-conjugative. Conjugative plasmids promote sexual conjugation between bacterial cells. Conjugative plasmids enable genetic transfer through conjugation. Non-conjugative plasmids can sometimes be co-transferred with conjugative ones. Different plasmids can coexist in a single bacterial cell if they're compatible.

5. Incompatible plasmids can be rapidly lost from the cell. The basis of plasmid incompatibility is not fully understood, but replication events play a role. Plasmid Classification Fertility or F plasmids promote conjugal transfer of plasmids Resistance or R plasmids provide resistance to antibacterial agents. Col plasmids code for colicins proteins that kill other bacteria.Example is ColE1 of E.coli . Degradative plasmids allow host bacterium to metabolize unusual molecules such as toluene and salicylic acid,example being TOL of Pseudomonas putida .

6. Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium tumefaciens ,which induce crown gall disease on dicotyledonous plants. Plasmids in organisms other than bacteria : Plasmids are not as common in organisms other than bacteria. The best characterized eukaryotic plasmid is the 2μm circle in Saccharomyces cerevisiae . The 2μm plasmid is important for gene cloning in yeast. The search for plasmids in other eukaryotes has been disappointing. .

7. Many higher organisms may not harbor plasmids within their cells. Bacteriophages: Basic features of bacteriophages Bacteriophages are viruses that specifically infect bacteria. They consist of a DNA or RNA molecule surrounded by a protein capsid. The infection process involves attachment, injection of DNA, replication, and Assembly of new phage particles. Some phages complete the infection cycle quickly, causing lysis of the bacterial cell.

8. This rapid infection cycle is known as the lytic cycle. Lysogenic phages: Lysogenic infection: Phage DNA retained in host bacterium for many cell divisions. Prophage : Integrated form of phage DNA in bacterial genome, remains quiescent. Lambda phage: Typical lysogenic phage, reverts to lytic mode and lyses cell. M13 phage: Continuous assembly and release of phage particles without cell lysis . Lambda and M13: Commonly used as cloning vectors due to their unique properties.

9. Gene organization in the lambda DNA molecule : Structure: DNA contained in the polyhedral head, tail attaches to bacterial surface and injects DNA into the cell. Lambda DNA molecule: 49 kb in size, extensively studied through gene mapping and DNA sequencing. Genetic map: Genes related in function are clustered together on the genome. Capsid genes: Grouped together in the left-hand third of the molecule. Prophage integration genes: Clustered in the middle of the molecule. Importance of clustering: Allows for coordinated gene expression and construction of lambda-based cloning vectors. The linear and circular forms of lambda DNA: The DNA molecule is linear with two free ends. It can form a circular, double-stranded molecule through base pairing of complementary single strands.

10. These single strands, called 'sticky' or cohesive ends, can join DNA molecules together. After the prophage leaves the host genome, new Lambda DNA molecules are made. The Lambda genomes are joined together at the cos sites. An enzyme cuts the joined Lambda genomes into individual ones at the cos sites. The enzyme also helps package the Lambda genomes into phage head structures. M13- a filamentous phage: M13 is a filamentous phage with a different structure from Lambda. It has a smaller, circular, single-stranded DNA molecule. M13 has fewer genes than lambda and simpler infection cycle. It doesn't need genes for insertion into the host genome. Inside the cell, M13 DNA acts as a template for synthesis of a complementary strand.

11. New M13 phage particles are continuously assembled and released. The attraction of M13 as a cloning vector M13 has a small genome size (<10 kb), making it an ideal vector. It can produce single-stranded DNA, which is useful for DNA sequencing and mutagenesis. Viruses as cloning vectors for other organisms: Mammalian viruses like SV40, adenoviruses, retroviruses, and insect baculoviruses have been extensively studied as cloning vectors.